AbstractBackground and ObjectivesWith the increasing use of fluorescence in medical applications, a comprehensive understanding of the effect of temperature on tissue autofluorescence is essential. The purpose of this study is to explore the effect of temperature on the fluorescence of porcine cornea and rat skin and determine the relative contributions of irreversible changes in optical properties and in fluorescence yield.Study Design/Materials and MethodsFluorescence, diffuse reflectance, and temperature measurements were acquired from excised porcine cornea and rat skin over a temperature range of 0–80°C. A dual excitation system was used with a 337 nm pulsed nitrogen laser for the fluorescence and a white light source for the diffuse reflectance measurements. A thermal camera measured tissue temperature. Optical property changes were inferred from diffuse reflectance measurements. The reversibility of the change in fluorescence was examined by acquiring measurements while the tissue sample cooled from the highest induced temperature to room temperature.ResultsThe fluorescence intensity decreased with increasing tissue temperature. This fluorescence change was reversible when the tissue was heated to a temperature of 45°C, but irreversible when heated to a temperature of 80°C.ConclusionAuto‐fluorescence intensity dependence on temperature appears to be a combination of temperature‐induced optical property changes and reduced fluorescence quantum yield due to changes in collagen structure. Temperature‐induced changes in measured fluorescence must be taken into consideration in applications where fluorescence is used to diagnose disease or guide therapy. Lasers Surg. Med. 44: 712–718, 2012. © 2012 Wiley Periodicals, Inc.